A method for treating dairy farm wastewater based on aspergillus, synechocystis and chlorella cascade treatment

Abstract

The application discloses a method for treating dairy farm wastewater based on Synechococcus, Aspergillus and green algae. In the wastewater treatment method, Aspergillus is used in the first step, and can realize rapid conversion of organic matter in dairy farm wastewater. Synechococcus is used in the second step, and has strong treatment capacity for high-concentration wastewater in a dairy farm, and is named Synechococcus ZSL-NS1. On the basis of the first two steps, the biomass can be rapidly increased. Finally, the removal rate of pollutants in the dairy farm wastewater can reach more than 80%, and the biomass yield is increased by 2.3 times. In addition, Synechococcus ZSL-NS1 can also produce chlorophyll, protein, polysaccharide, oil and other biomasses, so that the purpose of recycling is achieved while the pollution is removed, and the method has application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of aquaculture wastewater treatment and biotechnology, and in particular, relates to a method for treating dairy farm wastewater based on self-screening aspergillus, synechococcus and green algae in a step-by-step manner. BACKGROUND

[0002] Due to the special needs of milking halls, cow sheds and the like, dairy farms have become high-water-consumption industries, consuming a large amount of clean water resources while producing a large amount of high-concentration wastewater, which is complex in pollutant composition, high in concentration and difficult to degrade organic matter. If not effectively treated, it will cause secondary pollution to the surrounding environment.

[0003] Combining microalgae cultivation with wastewater treatment has become a research hotspot in the field of water treatment. This method takes advantage of the growth of microalgae to convert the nutrients in wastewater into microalgae biomass and achieve purification and treatment of wastewater resources. At present, this method has gradually been recognized and applied in the field of environmental protection. However, there are still many problems in this technology, which limit its industrial application. For example, the composition of dairy farm wastewater is complex, the concentration is high, and the light transmittance is poor, which all have inhibitory effects on the growth and reproduction of microalgae and affect their purification effect on wastewater. In addition, different microalgae have different tolerances to wastewater and different available nutrient compositions, so it is difficult for traditional commercially available microalgae to be directly used for dairy farm wastewater treatment. SUMMARY

[0004] In view of the problems existing in the current microalgae wastewater treatment technology, the present application provides a method for treating dairy farm wastewater based on self-screening aspergillus, synechococcus and green algae in a step-by-step manner according to the characteristics of dairy farm wastewater and the physiological characteristics of different microalgae. By adding self-screening filamentous fungi in the first step, the rapid conversion of organic matter in wastewater is realized, laying a foundation for the growth and utilization of microalgae. By adding self-screening synechococcus in the second step, the rapid purification of high-concentration wastewater is realized. By adding commercially available green algae / bryozoan in the third step, the further purification of wastewater and the large-scale value-added of biomass are realized. Through the above, efficient purification of dairy farm wastewater and efficient improvement of biomass yield can be realized.

[0005] The technical problem of the present application is solved by adopting the following technical solution:

[0006] A method for treating dairy farm wastewater based on self-screening aspergillus, synechococcus and green algae in a step-by-step manner, the specific steps comprising the following:

[0007] S1: Step-by-step wastewater treatment method: centrifuge and concentrate the synechococcus ZSL-NS1, chlorella and bryozoan to the logarithmic phase after being cultured in a light incubator using BG11 culture solution as seed liquid; store the aspergillus culture to the recombination amount for later use;

[0008] S2: Aspergillus is added to the dairy farm oxidation pond wastewater at 0.3 g / L, the cultivation conditions are about 20-30 DEG C, natural light source, and the treatment is performed for 2-5 days in total;

[0009] S3: The treatment liquid in S1 is centrifuged at 7000-8000 r / min, and the supernatant is collected; Synechocystis is added to the supernatant at an amount of 0.2 g / L, and is shaken uniformly, the light intensity is 4000-5000 Lux, the light-dark ratio is 12h:12h, the temperature is 20-25 DEG C, and the treatment is performed for 4 days in total;

[0010] S4: The treatment liquid in S2 is centrifuged at 7000-8000 r / min, and the supernatant is collected; Chlorella or Euglena is added to the supernatant at an amount of 0.2-0.5 g / L, and is shaken uniformly, the light intensity is 3000-4000 Lux, the light-dark ratio is 12h:12h, the temperature is 25 DEG C, the treatment is performed for 6 days in total, the treatment is performed in a shaking bed mode to avoid microalgae sedimentation or wall sticking, 100-200 rpm, 30-60 min, 3-5 times a day;

[0011] S4: Wastewater simultaneous treatment method: the three kinds of microorganisms in S1 are simultaneously added to the wastewater, and are reacted for 12-15 days, the light intensity is 3000-4000 Lux, the light-dark ratio is 12h:12h, the temperature is 25 DEG C, and the treatment is performed for 12 days in total, wherein the natural light is 0-2 days.

[0012] Moreover, the treatment in step S3 is performed in a shaking bed mode to avoid microalgae sedimentation or wall sticking, 100 rpm, 30 min, 3 times a day.

[0013] Moreover, the algal strain of Synechocystis sp. ZSL-NS1 (Svnechocvstis sp. ZSL-NS1) has been preserved in the China Center for Type Culture Collection on November 07, 2024, and the preservation number is CCTCC M 20242468.

[0014] Moreover, the Chlorella is Chlorella FACHB-2338, and the Euglena is Euglena FACHB-848.

[0015] Moreover, the preservation number of Aspergillus is CGMCC No 3.15297.

[0016] The advantages and positive effects of the present application are:

[0017] The present application screens a strain of strong pollution-resistant Synechococcus ZSL-NS1 from biogas pool wastewater of a dairy farm, and then combines a commercially available good effect Aspergillus and Chlorella / Euglena to treat the wastewater of the dairy farm by using the "filamentous fungi"-"cyanobacteria"-"green algae" in stages: in the first step, the self-screened Aspergillus can realize the conversion of organic matter in the wastewater to small molecules, providing a nutrient basis for the following steps; in the second step, the high tolerance of the self-screened Synechococcus ZSL-NS1 can overcome the growth inhibition problem caused by high-concentration wastewater, and further realize the reduction of wastewater concentration and the conversion of organic matter; in the third step, the use of Chlorella / Euglena can further purify the wastewater and realize the accumulation of a large amount of microalgae biomass.

[0018] After the three-stage treatment method of the present application, the dairy wastewater can be effectively purified, and the removal rate of pollutants therein reaches more than 80%; in addition, high-value microalgae biomass can be obtained, and the cumulative amount of the biomass is increased by 2.3 times, and the content of protein in the biomass is more than 50%.

[0019] The method in the present application can effectively overcome the problems of poor tolerance, growth inhibition and low biomass yield of microorganisms / microalgae in traditional methods, and achieve the dual purposes of pollution reduction and carbon reduction, cost reduction and efficiency improvement. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a microscope morphological photo of Synechococcus.

[0021] Figure 2 It is a Neighbor-Joining phylogenetic tree of strain NS1 based on 16S rRNA gene sequence alignment results with Pseudomonas aeruginosa DSMZ50071 (X06684) as the outgroup.

[0022] Figure 3 It is a microscope morphological photo of Aspergillus.

[0023] Figure 4 It is the treatment effect of the step-by-step treatment method on wastewater.

[0024] Figure 5 It is the dry weight change of microalgae biomass in different treatments (T1: Synechococcus ZSL-NS1; T2: Chlorella; T3: Euglena; T4: Synechococcus + Chlorella; T5: Synechococcus + Euglena).

[0025] Figure 6-1 Paper Figure 6-3 It is the composition of microalgae and the yield of extracellular polymeric substances (EPS). DETAILED DESCRIPTION

[0026] The above content of the present application will be further illustrated in detail by way of examples below, but it should not be understood that the above subject matter of the present application is limited to the following examples only, and any technology realized based on the above content of the present application falls within the scope of the present application.

[0027] In specific examples, the dairy farm wastewater in the treatment effect experiment of the present application is wastewater from an oxidation pond, an anaerobic biogas tank or a stabilization pond of a certain dairy farm in Tianjin; the dairy farm wastewater used for screening cyanobacteria is effluent from a biogas tank of a certain dairy farm in Tianjin.

[0028] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0029] Example 1

[0030] The present application isolated a Synechocystis sp. from the soil beside a manure storage tank of a certain dairy farm in Tianjin in 2023, which has a good survival condition and obvious decontamination effect in dairy farm wastewater. It is obtained by screening in dairy farm biogas tank wastewater, and has strong treatment capacity for high-concentration dairy farm wastewater. The strain of Synechocystis sp. named ZSL-NS1 has been preserved in China Center for Type Culture Collection on November 07, 2024, and the preservation number is CCTCC NO. M 20242468. The following methods for isolation and cultivation of Synechocystis ZSL-NS1 are described in detail as follows:

[0031] The wastewater sample was gradient diluted, and then plated. Single algae colonies were picked and inoculated into growth medium. After 15 days of culture at 25℃, 3000 Lux light, and 16:8 light-dark cycle, blue-green algae liquid was obtained. The growth condition and morphology were observed under a microscope, and the culture was purified several times. After identification, it was Synechocystis ZSL-NS1. After obtaining the single algae strain liquid, it was expanded until enough algae liquid (the volume ratio during expansion: algae liquid: medium = 1:5).

[0032] The formula of growth medium (BG11) is as follows: sodium nitrate 1.5 g, potassium phosphate dibasic 0.04 g, magnesium sulfate heptahydrate 0.075 g, calcium chloride dihydrate 0.036 g, ferric ammonium citrate 0.006 g, citric acid 0.006 g, ethylenediaminetetraacetic acid disodium salt 0.001 g, sodium carbonate 0.02 g, trace element stock solution 1 mL, water to 1000 mL, pH adjusted to about 7.1, and autoclaved at 121℃ for 20 min.

[0033] The formula of the trace element mother liquor is: boric acid 2.86 g / L, manganese chloride tetrahydrate 1.86 g / L, zinc sulfate heptahydrate 0.22 g / L, copper sulfate pentahydrate 0.08 g / L, sodium molybdate dihydrate 0.39 g / L, cobalt nitrate hexahydrate 0.05 g / L, and water to 1000 mL.

[0034] The results show that Synechococcus ZSL-NS1 is blue-green, the cells are spherical or oval, the cells are small, and the diameter is generally about 1-5 microns. The cell surface is relatively smooth, and there is no obvious protrusion or accessory. In some cases, Synechococcus cells can gather to form groups, and the groups have various morphologies. The cells in the groups are arranged relatively closely.

[0035] Example 2

[0036] Comparison test of treatment effect of dairy farm wastewater

[0037] S1: Ladder type wastewater treatment method: Synechococcus ZSL-NS1, Chlorella (FACHB-2338) and Euglena (FACHB-848) were cultured in a light incubator using BG11 culture solution to the logarithmic phase, then centrifuged and concentrated as seed liquid; Aspergillus sp. (CGMCC No 3.15297) (CN109170279A) was cultured to the recombination amount and stored for standby.

[0038] S2: Aspergillus sp. (CGMCC No 3.15297) (CN109170279A) was added to the dairy farm oxidation pond wastewater at 0.3 g / L, and the incubation conditions were about 25℃, natural light source, and a total treatment time of 2 days.

[0039] S3: The treatment liquid in S1 was centrifuged at 7000-8000 r / min for 10 minutes, and the supernatant was collected; Synechococcus was added to the supernatant at a concentration of 0.2 g / L, shaken uniformly, the light intensity was 4000-5000 Lux, the light-dark ratio was 12h:12h, the temperature was 25℃, and the total treatment time was 4 days.

[0040] S4: The treatment liquid in S2 was centrifuged at 7000-8000 r / min for 10 minutes, and the supernatant was collected; Chlorella or Euglena was added to the supernatant at a concentration of 0.2 g / L, shaken uniformly, the light intensity was 3000-4000 Lux, the light-dark ratio was 12h:12h, the temperature was 25℃, and the total treatment time was 6 days. The treatment was carried out in a shaking bed to prevent the microalgae from settling or sticking to the wall, at 100 rpm, 30 minutes, 3 times a day.

[0041] S4: Simultaneous wastewater treatment method: the three microorganisms in (1) were added to the wastewater at the same time, and reacted for 12 days, the light intensity was 3000-4000 Lux, the light-dark ratio was 12h:12h, the temperature was 25℃, and the total treatment time was 12 days, of which the natural light was 0-2 days.

[0042] Example 3

[0043] Microalgae biomass production experiment

[0044] The microalgae samples were collected and analyzed for dry weight using the dry weight method as in (1) of Example 3. Figure 5 ).

[0045] Example 4

[0046] Microalgae composition experiment test

[0047] The microalgae samples were collected and analyzed for the composition of the microalgae and the production of extracellular polymeric substances (EPS) as in (1) of Example 3.

[0048] The above description is only the preferred embodiment of the present application, not any form of limitation to the present application, any skilled in the art, without departing from the technical solution of the present application, according to the technical essence of the present application, any simple modification, equivalent replacement and improvement of the above examples, etc., still belong to the protection scope of the technical solution of the present application.

[0049] Although the embodiments of the present application are disclosed for the purpose of illustration, those skilled in the art can understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, therefore, the scope of the present application is not limited to the disclosed content of the embodiments.

Claims

1. A method for treating dairy farm wastewater in a stepped manner based on Synechocystis, Aspergillus, and Chlorella, characterized in that: The specific steps include the following: S1: Stepwise wastewater treatment method: Synechocystis ZSL-NS1, Chlorella vulgaris and Euglena spp. are cultured in a light incubator with BG11 culture medium to the logarithmic phase and then concentrated by centrifugation to obtain seed culture; Aspergillus is cultured to the recombinant amount and then stored for later use; S2: Add Aspergillus at a concentration of 0.3 g / L to the oxidation pond wastewater of the dairy farm. The cultivation conditions are 20-30℃, natural light, and a total treatment period of 2-5 days. S3: Centrifuge the treatment liquid from S1 at 7000-8000 r / min and collect the supernatant; add Synechocystis at a concentration of 0.2 g / L to the supernatant, shake well, apply light at an intensity of 4000-5000 Lux, a light-dark ratio of 12 h:12 h, and a temperature of 20-25 ℃ for a total of 4 days. S4: Centrifuge the treatment liquid from S2 at 7000-8000 r / min and collect the supernatant; add Chlorella or Euglena at a concentration of 0.2-0.5 g / L to the supernatant, shake well, maintain a light intensity of 3000-4000 Lux, a light-dark ratio of 12 h:12 h, and a temperature of 25℃ for a total of 6 days. Use a shaking table to avoid microalgae precipitation or adhesion to the wall, at 100-200 rpm for 30-60 min, 3-5 times a day. S4: Simultaneous wastewater treatment method: The three types of microorganisms in S1 are added to the wastewater simultaneously and reacted for 12-15 days. The light intensity is 3000-4000 Lux, the light-dark ratio is 12h:12h, the temperature is 25℃, and the treatment lasts for a total of 12 days, including 0-2 days under natural light. The *Syntrophus synechocystis* strain ZSL-NS1 was deposited at the China Center for Type Culture Collection (CCTCC) on November 7, 2024, with accession number CCTCC NO.M 20242468, at Wuhan University, Wuhan, China.

2. The method for treating dairy farm wastewater in a stepped manner based on Aspergillus, Synechocystis, and Chlorella according to claim 1, characterized in that: In step S3, the treatment uses a shaking table to prevent microalgae from settling or sticking to the wall, at 100 rpm for 30 minutes, three times a day.

3. The method for treating dairy farm wastewater in a stepped manner based on Aspergillus, Synechocystis, and Chlorella according to claim 1, characterized in that: The Chlorella species mentioned are Chlorella FACHB-2338 and Euglena species are Euglena FACHB-848.

4. The method for treating dairy farm wastewater in a stepped manner based on Aspergillus, Synechocystis, and Chlorella according to claim 1, characterized in that: The accession number for Aspergillus is CGMCC No. 3.15297.

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